antioxidants Review Pharmacological Protection against Ischemia-Reperfusion Injury by Regulating the Nrf2-Keap1-ARE Signaling Pathway Bercis Imge Ucar 1,* , Gulberk Ucar 2 , Sarmistha Saha 3 , Brigitta Buttari 3 , Elisabetta Profumo 3 and Luciano Saso 4 1 Department of Biochemistry, Faculty of Medicine, Hacettepe University, 06100 Ankara, Turkey 2 Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey; [email protected] 3 Department of Cardiovascular, Endocrine-Metabolic Diseases, and Aging, Italian National Institute of Health, 00161 Rome, Italy; [email protected] (S.S.); [email protected] (B.B.); [email protected] (E.P.) 4 Department of Physiology and Pharmacology “Vittorio Erspamer” Sapienza University, 00161 Rome, Italy; [email protected] * Correspondence: [email protected] Abstract: Ischemia/reperfusion (I/R) injury is associated with substantial clinical implications, in- cluding a wide range of organs such as the brain, kidneys, lungs, heart, and many others. I/R injury (IRI) occurs due to the tissue injury following the reestablishment of blood supply to ischemic tissues, leading to enhanced aseptic inflammation and stimulation of oxidative stress via reactive oxygen and nitrogen species (ROS/RNS). Since ROS causes membrane lipids’ peroxidation, triggers loss of membrane integrity, denaturation of proteins, DNA damage, and cell death, oxidative stress plays a critical part in I/R pathogenesis. Therefore, ROS regulation could be a promising therapeutic Citation: Ucar, B.I.; Ucar, G.; Saha, S.; strategy for IRI. In this context, Nrf2 (NF-E2-related factor 2) is a transcription factor that regulates Buttari, B.; Profumo, E.; Saso, L. the expression of several factors involved in the cellular defense against oxidative stress and in- Pharmacological Protection against flammation, including heme oxygenase-1 (HO-1). Numerous studies have shown the potential role Ischemia-Reperfusion Injury by of the Nrf2/HO-1 pathway in IRI; thus, we will review the molecular aspects of Nrf2/Kelch-like Regulating the Nrf2-Keap1-ARE ECH-associated protein 1 (Keap1)/antioxidant response element (ARE) signaling pathway in I/R, Signaling Pathway. Antioxidants 2021, and we will also highlight the recent insights into targeting this pathway as a promising therapeutic 10, 823. https://doi.org/10.3390/ antiox10060823 strategy for preventing IRI. Academic Editor: Tommaso Angelone Keywords: Nrf2/Keap1/ARE signaling pathway; ischemia-reperfusion injury; Nrf2 activators Received: 7 April 2021 Accepted: 15 May 2021 Published: 21 May 2021 1. Introduction 1.1. Ischemia/Reperfusion Injury (IRI) Publisher’s Note: MDPI stays neutral IRI injury is a sequential catastrophic event with high morbidity and mortality, usually with regard to jurisdictional claims in seen after thoracoabdominal surgeries, cardiopulmonary bypass, hemorrhagic, traumatic, published maps and institutional affil- or septic shock, severe burns, or after transplantation operations [1] characterized by loss of iations. blood flow, causing hypoxia and lack of nutrition (ischemia phase) followed by restoration of the blood supply (reperfusion phase), which often results in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) [2–6]. Inter- ruption of the blood flow causes reduced oxygen-dependent-ATP production, impaired Copyright: © 2021 by the authors. ion-pump activities, hypoxanthine and lactate accumulation (acidosis), intracellular cal- Licensee MDPI, Basel, Switzerland. cium overload, which triggers reactive oxygen species (ROS) production. Reintroducing This article is an open access article oxygen to the ischemic tissue contributes to the formation of oxidant-dependent pro- distributed under the terms and inflammatory mediators, increase in calcium overload in the cell. It also activates calpains conditions of the Creative Commons leading to programmed cell death, ROS production from previously sequestered hypoxan- Attribution (CC BY) license (https:// thine consequent to reoxygenation, thereby results in a positive feedback loop by opening creativecommons.org/licenses/by/ the mitochondrial permeability transition pores (mPTP) to produce more ROS. Eventu- 4.0/). Antioxidants 2021, 10, 823. https://doi.org/10.3390/antiox10060823 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 823 2 of 19 ally, excess amounts of ROS result in oxidative stress and oxidase proteins, lipids, and DNA [6,7]. 1.2. Oxidative Stress Oxidative stress is a pathological process caused by the imbalance between redox systems in favor of the oxidant systems. This imbalance usually originates either from the excessive production of ROS or the diminished ROS scavenging capacity [3–5]. ROS and reactive nitrogen species (RNS) can be sub-grouped into free radicals like superoxide •− (O2 ), oxygen, hydroxyl, and peroxyl radicals, nitric oxide and nitrogen dioxide, and non-radicals such as hydrogen peroxide (H2O2), hypochlorous acid, singlet oxygen, nitrous acid, nitroxyl anion, lipid peroxides’ aldehydes, and peroxynitrite [3–5,8–11]. These can be introduced from exogenous sources like environmental pollutants [8–10], ultraviolet irradiation [9,10], carcinogens [9,10], and cigarette smoke [9,10], produced on purpose for physiological signal transduction of cells such as inflammation, integrin recruitment, growth factor signaling (for instance, correct interaction between insulin, epidermal growth factor, platelet-derived growth factor, and their receptors), adhesion to the extracellular matrix, or can be a metabolic by-product [3–5,8–11]. The xanthine oxidase system, NADPH oxidase system, mitochondrial electron transport chain, uncoupled nitric oxide synthase system, and non-enzymatic sources (like hemoglobin and myoglobin) generate oxidative stress [5,8]. 1.3. Pro-Inflammatory Features of IRI Tissue destruction can be seen under excessive ROS exposure via lipid peroxidation in the cell membrane and organelle lipids, DNA oxidation, activation of matrix metallopro- teinases, induced osmotic cell lysis, increased mitochondrial permeability, or via indirect mechanisms including free radical formation by the interaction with molecular oxygen, NO, free iron, or fatty acids or free iron. These mechanisms enhance the formation of oxidant-dependent pro-inflammatory mediators and upregulation of cytokine/chemokine and adhesion molecule expression during reperfusion [7]. Indeed, although the mecha- nism of IRI is a form of pathogen-free inflammation, it displays a series of inflammatory reactions resembling infectious processes such as inducing cytokine and chemokine pro- duction with infiltration of immune system cells [3,4]. After reperfusion, the reciprocity between the innate and adaptive immune cells is critical for tissue damage induction. Neu- trophils and monocytes become activated by many damage-associated molecular patterns (DAMPs) released by necrotic cells, which in turn polarize T lymphocytes toward the Th1 pro-inflammatory phenotype. The primary immune response expands tissue-associated damage by inducing NLRP3-inflammasome, TLR9, and the NET formation [12,13]. Consequently, the myeloid differentiation primary response gene 88 (MyD88) and nuclear factor-κB (NF-κB) pathways are activated, followed by initiation of the inflam- matory mediator release [14], which amplifies the aforementioned immune cells. NLRP3 inflammasome activation also generates caspase-mediated cell death [15]. From a wound- healing perspective, after the inflammatory phase dominated by Th1 polarized cells, a second, anti-inflammatory reparative phase leading to wound healing and scar formation comes later. Cytokines have a crucial role in tissue repair. IL-6, IL-10, transforming growth factor-beta (TGF-β), and a subpopulation of T-lymphocytes known as ‘T Regulatory cells’ (Treg) have all been associated with suppressing the pro-inflammatory response and steer- ing the immune system towards repair and resolution following I/R [16,17]. Apoptotic neutrophils induce an M2 phenotype in infiltrated macrophages upon their phagocytosis, inhibiting the macrophage pro-inflammatory tissue-damaging response and leading them to produce IL-10 and TGF-β [18,19]. Notably, IL-10 may serve to dampen both Th1 and Th2 inflammation, thus inhibiting I/R-derived damage, as well as excessive tissue scarring during the reparative phase. Accumulation of immune and inflammatory cells causes ROS to pile up and fibrosis by inducing fibroproliferation and abnormal collagen accumulation, as well as inflammation-mediated tissue destruction. Additionally, endothelial swelling Antioxidants 2021, 10, 823 3 of 19 and glycocalyx degradation in I/R lead to loss of intercellular contact between endothelial cells, consequently causing various vascular abnormalities, including elevated vascular permeability and leakage, dysregulated vasodilation and vasoconstriction, dysfunction of the endothelial cell barrier, inflamed endothelium, along with activation of complement and coagulation systems [3]. Other manifestations of I/R with similar pro-inflammatory features include cell death via necrosis or programmed cell death (apoptosis and autophagy). In normal conditions, autophagy recycles damage proteins and organelles after lysosomal degradation as a cytoprotective mechanism. However, ROS accumulation during I/R surpasses autophagic clearance capacity and eventually leads to cell death [3,6]. It has already been known that the disproportionate generation of ROS is the crucial